1. Field of the Invention
The present invention relates to DNA-immobilized microspheres used, for example, for purifying a protein. More particularly, it relates to DNA-immobilized microspheres comprising DNA chains having base sequences capable specifically of binding a transcription-controlling factor protein, and a carrier which does not adsorb any protein, the DNA chains and the carrier being chemically bound to each other. Said DNA-immobilized microspheres are used in a process for purifying a transcription-controlling factor which comprises adsorbing the transcription-controlling factor on the DNA chains having base sequences capable specifically of binding the transcription-controlling factor protein, removing other proteins, and then releasing the transcription-controlling factor from the DNA chains.
2. Related Art
One of the main branches of genetic engineering which has been greatly advanced in recent years is analysis of genes themselves. In an analysis, analysis of transcription-controlling factors leads to elucidation of the mechanism of transcriptional control in genes and hence is important.
Transcription is a process of synthesis of RNA in accordance with DNA as gene by RNA polymerase and is divided into initiation, elongation and termination steps.
In the transcription initiation step, RNA polymerase binds to a specific base sequence of DNA which is called "promoter", to initiate transcription. It is considered that in the promoter of procaryotic cells, a base sequence called "Pribnow box" which is located about 10 base pairs upstream to the transcription initiation site (this base sequence is basically TATAAT though somewhat different depending on promoter), is a site to which RNA polymerase actually binds. A certain protein binds to a specific base sequence called "operator" to accelerate or suppress the transcription from the promoter. In the case of eucaryotic cells, it is suggested that a base sequence called "TATA box" or "Goldberg-Hogness box" which is located 20 to 30 base pairs upstream to the transcription initiation site [this base sequence is basically TATA.sub.T.sup.A A.sub.T.sup.A (.sub.T.sup.A represents A or T) though somewhat different depending on promoter], determines the initiation of transcription. A factor is separated and binds to the TATA box to control the transcription. As to the control of the amount of the transcription, it is considered that there exists a characteristic, a specific base sequence upstream to TATA box, which participates in the transcriptional control of each gene. In addition, there is a specific base sequence called "enhancer" which enhances the transcriptional efficiency. Three kinds of RNA polymerases are present in eucaryotic cells: RNA polymerase II participates in the usual synthesis of a mRNA precursor, RNA polymerase I in synthesis of ribosomal RNA, and RNA polymerase III in synthesis of 5S RNA and tRNA. For each of these syntheses, there exist regulatory genes specific for the transcription for the synthesis.
In the transcription elongation step, in the case of procaryotic cells, a sequence called "attenuator" exists as a transcription termination signal inside an operon and controls the gene expression. In the case of animal cells, substantially no regulatory signal corresponding to an attenuator have been reported, but it has been suggested that such a regulatory signal is present in the late gene region of Simian Virus 40.
In the transcription termination step, in the case of procaryotic cells, there exist base sequences called "terminators" which order termination of transcription. The terminators of Escherichia coli are roughly divided into those requiring a protein factor called ".rho. factor" (.rho.-dependent terminators) and those requiring no .rho. factor (.rho.-independent terminators). The terminators are complicatedly controlled by participation of .lambda.N anti-termination protein or the like. In the case of eucaryotic cells, the transcription termination step has been investigated but has not yet been elucidated.
Thus, a large number of signals in a gene participate in transcription. Many of the signals function by interaction with transcription-controlling factors which are proteins. Elucidation and utilization of the mechanism of such transcription will lead to great progress, for example, in control of biogenic actions and production of useful materials by genetic engineering, and are of important significance in the fields of medicines, fermentation, etc.
For analysis of the important transcription-controlling factors in a gene, it is necessary to separate and purify a protein which acts as the controlling factor. But, such a transcription-controlling factor protein only exist in very small amounts and hence is difficult to purify. A transcription-controlling factor protein which binds directly to a gene is separated and purified by affinity chromatography using DNA chains containing binding sites, as a ligand, and gel particles of cellulose, agarose, etc. have been used therefor. However, most of these gels on the market have a particle size of more than several tens of micrometers, a small total area of particle surface, and a wide particle size distribution, and hence cannot be said to be sufficient in efficiency, reproducibility, etc. when used for the purpose described above. Furthermore, when the gel particles are packed in a column, a solution cannot be passed through the column at a high pressure from the viewpoint of the strength of the gel particles because the gel particles contain water at more than several tens of times as much as the gel particles. Therefore they are not efficient.